Induction of STAT1 Phosphorylation at Serine 727 and Expression of Proinflammatory Cytokines by Porcine Reproductive and Respiratory Syndrome Virus

Zhang Y (2013) Induction of STAT1 Phosphorylation at Serine 727 and Expression of Proinflammatory Cytokines by Porcine Reproductive and Respiratory Syndrome Virus. PLoS ONE 8(4): e61967. doi:10.1371/journal.pone.0061967 Induction of STAT1 Phosphorylation at Serine 727 and Expression of Proinflammatory Cytokines by Porcine Reproductive and Respiratory Syndrome Virus Ying Yu 0 Rong Wang 0 Yuchen Nan 0 Linsheng Zhang 0 Yanjin Zhang 0 Jianming Qiu, University of Kansas Medical Center, United States of America 0 1 College of Life Sciences, Northwest A&F University , Yangling, Shaanxi, China, 2 Molecular Virology Laboratory , VA-MD Regional College of Veterinary Medicine and Maryland Pathogen Research Institute, University of Maryland , College Park, Maryland , United States of America Porcine reproductive and respiratory syndrome virus (PRRSV) is a viral pathogen that causes acute respiratory illnesses in young pigs. Since 1987, PRRSV has contributed substantial economic losses to the swine industry. Elevation of proinflammatory cytokines in PRRSV-infected pigs is thought to contribute to PRRSV pathogenesis. In this study, PRRSV VR2385, a Type 2 strain with moderate virulence, was found to induce phosphorylation of signal transducer and activator of transcription 1 (STAT1) at serine 727 (pSTAT1-S727) in MARC-145 cells. No phosphorylated STAT1 at tyrosine 701 was detected, which indicates that the pSTAT1-S727 elevation was interferon-independent. The PRRSV-induced pSTAT1-S727, however, was dose-dependent and its levels increased with infection time. IngelVac PRRS MLV strain had a minimal effect on pSTAT1-S727. Compared to MLV-infected cells, VR-2385 infection caused significantly higher level of expression of proinflammatory cytokines, including interleukin 1 beta (IL-1beta) and IL-8. The VR-2385-induced pSTAT1-S727 and cytokine expression were reduced after SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), or methylthioadenosine (MTA), a methyl transferase inhibitor, was added to the cells. The SB203580 and MTA-mediated inhibition suggested that the virus-induced pSTAT1-S727 was dependent on p38 MAPK pathway. In primary porcine alveolar macrophages (PAMs), VR-2385 also induced pSTAT1-S727 and expression of proinflammatory cytokines and chemokines, including IL-1beta, IL-8, chemokine ligand 2 (CCL2) and chemokine (C-X-C motif) ligand 10 (CXCL10). Similarly, SB203580 treatment of PAM cells blocked the elevation of pSTAT1-S727 and cytokine expression. Overexpression of individual viral proteins showed that non-structural protein 12 (nsp12) was able to induce elevation of pSTAT1-S727 and the expression of IL-1b and IL-8. These results indicated that PRRSV VR-2385 induces pSTAT1-S727 and the expression of proinflammatory cytokines, which contributes to the insight of PRRSV pathogenesis. - Funding: This study was partially supported by National Pork Board. Y. Yu, R. Wang, and Y. Nan were partially supported by China Scholarship Council. No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Porcine reproductive and respiratory syndrome (PRRS) is a widespread viral disease that has caused substantial economic losses to the swine industry [1]. The disease remains a major challenge since it was first reported in the United States in 1987. Moreover, outbreaks of highly pathogenic PRRS in Asia in recent years [2,3] raise further concerns. The causative agent of PRRS is the PRRS virus (PRRSV), an enveloped, positive-sense, singlestrand RNA virus belonging to the genus Arterivirus, family Arteriviridae, order Nidovirales [4]. The entire genome of PRRSV is approximately 15 kb including ten open reading frames (ORFs). The ORF1a and ORF1b encode pp1a and pp1ab polyproteins, which are cleaved into fourteen non-structural proteins [5,6]. In addition to the two large ORFs, PRRSV genome contains the other eight ORFs: 2, 2a, 3, 4, 5, 5a, 6 and 7 that encode eight structural proteins: GP2, E, GP3, GP4, GP5, 5a, M and N, respectively [710]. PRRSV propagation in vitro relies on an epithelial-derived monkey kidney cell line, MARC-145 [11], and primary cultures of porcine pulmonary alveolar macrophages (PAMs). PAMs are the primary target cells for PRRSV during its acute infection in pigs [12]. Many efforts to control PRRS, including attenuated live virus vaccines, have been tested, but few are successful because of the antigenic and genomic diversity among PRRSV isolates, as well as the persistence of the virus in infected herds. PRRSV causes acute phase response in pigs by replicating in the lungs and lymphoid organs. Up-regulated proinflammatory cytokines, such as interleukin 1 (IL-1), IL-6 and tumor necrosis factor alpha (TNF-a), initiate this acute phase response and relate to intrinsic pathogenicity in the respiratory infection [1315]. Expression of these cytokines correlates with the severity of pulmonary pathology and the number of macrophages in lung lesion [16]. Evaluation of early cytokine responses to PRRSV infection showed that three serum cytokines IL-8, IL-1b, and IFNc were correlated with virus level in pigs [17]. These studies have shown the importance of proinflammatory cytokines in PRRSV infection, but the induction of these genes in PRRSV infection is not well-defined. In this study, a moderate virulent strain VR-2385 was found to induce phosphorylation of STAT1 (signal transducer and activator of transcription 1) at serine 727 (pSTAT1-S727) in MARC-145 and PAM cells. The virus infection increased expression of some proinflammatory cytokines, including IL-1b and IL-8. Inhibition of p38 mitogen-activated protein kinase (MAPK) blocked elevation of pSTAT1-S727 and expression of the cytokine genes in VR2385-infected cells. Overexpression of individual viral proteins showed that nsp12 was possibly responsible for the upregulation of pSTAT1-S727. PRRSV Infection of MARC-145 Cells Induces Phosphorylation of STAT1 Serine 727 During our study of PRRSV inhibition of interferon-activated signaling, we noticed that PRRSV VR-2385 induced the elevation of phosphorylated STAT1 at serine 727. STAT1 is an essential transcription factor for the expression of the majority of IFNinduced genes [18,19]. MARC-145 cells were inoculated with two different Type 2 PRRSV strains, VR-2385, a moderate virulent strain, and MLV, an avirulent strain, both at 1 multiplicity of infection (MOI). Mock-infected cells were included as controls. The level of pSTAT1-S727 in VR-2385-infected cells 24 h post infection (hpi) was considerably increased in comparison to the mock-infected cells (Fig. 1A). The MLV infection had a minimal effect on pSTAT1-S727 level. Densitometry analysis showed that the pSTAT1-S727 level in VR-2385-infected cells was 2.7-fold higher than that of the mock (...truncated)